Color, activity period, and eye structure in four lineages of ants: Pale, nocturnal species have evolved larger eyes and larger facets than their dark, diurnal congeners

The eyes of insects display an incredible diversity of adaptations to enhance vision across the gamut of light levels that they experience. One commonly studied contrast is the difference in eye structure between nocturnal and diurnal species, with nocturnal species typically having features that enhance eye sensitivity such as larger eyes, larger eye facets, and larger ocelli. In this study, we compared eye structure between workers of closely related nocturnal and diurnal above ground foraging ant species (Hymenoptera: Formicidae) in four genera (Myrmecocystus, Aphaenogaster, Temnothorax, Veromessor). In all four genera, nocturnal species tend to have little cuticular pigment (pale), while diurnal species are heavily pigmented (dark), hence we could use cuticle coloration as a surrogate for activity pattern. Across three genera (Myrmecocystus, Aphaenogaster, Temnothorax), pale species, as expected for nocturnally active animals, had larger eyes, larger facet diameters, and larger visual spans compared to their dark, more day active congeners. This same pattern occurred for one pale species of Veromessor, but not the other. There were no consistent differences between nocturnal and diurnal species in interommatidial angles and eye parameters both within and among genera. Hence, the evolution of eye features that enhance sensitivity in low light levels do not appear to have consistent correlated effects on features related to visual acuity. A survey across several additional ant genera found numerous other pale species with enlarged eyes, suggesting these traits evolved multiple times within and across genera. We also compared the size of the anterior ocellus in workers of pale versus dark species of Myrmecocystus. In species with larger workers, the anterior ocellus was smaller in pale than in dark species, but this difference mostly disappeared for species with smaller workers. Presence of the anterior ocellus also was size-dependent in the two largest pale species.

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Unfunded studies
Enter: The author(s) received no specific funding for this work.  If the data are held or will be held in a public repository, include URLs, accession numbers or DOIs. If this information will only be available after acceptance, indicate this by ticking the box below. For example: All XXX files are available from the XXX database (accession number(s) XXX, XXX. Broadly, we were interested in the evolution and consequences of these associations, and 38 we used a comparative approach to examine these relationships in four ant genera in two 39 subfamilies (Myrmecocystus -subfamily Formicinae and Aphaenogaster, Temnothorax, 40 Veromessor -subfamily Myrmicinae) that contain pale and dark species. This multitaxa 41 approach strengthened our ability to make evolutionary inferences. 42 We first quantified for each genus the association between cuticular pigmentation and 43 daily activity patterns, i.e., whether pale species are more nocturnal than their dark congeners. 44 We then examined how eye size varies with body color and activity time. Specifically, we 45

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All brightness, body size, and eye measurements were made from photographs of 125 workers as described below. We quantified worker color using the brightness value (v or B, in 126 HSV format), which is similar to the lightness value in HSL that has been used to characterize 127 body color in other studies of ants [8, 10]. Brightness (B) was measured using the color window 128 in Adobe Photoshop from photographs downloaded from Antweb (www.antweb.org). 129 Obviously discolored specimens were excluded, i.e., those in which the color differed 130 substantially from intraspecific specimens recently collected by RAJ. Using the photograph of 131 the worker body in profile, we measured B on the head (immediately posterior to the eye), 132 mesosoma (center of mesopleura), and gaster (anteroposterior portion of first gastral tergum), 133 then averaged these values for each worker, then averaged that value across all workers for each 134 species. We compared mean B values for pale versus dark species using a t-test. 135 136 137 Activity patterns 138 The relationship between color and activity pattern was evaluated by gleaning above-139 ground foraging times from literature, personal observations, and personal communications. 140 Foraging times were classified as one or more of the following: diurnal, nocturnal, matinal, 141 crepuscular, and variable. The category "variable" included species in which foraging time 142 varies seasonally with temperaturediurnal when days are cool, crepuscular-matinal as 143 temperatures increase, and nocturnal when nights are warm. Exclusively day-active or night-144 active species were classified as diurnal and nocturnal, respectively. Species that forage during 145 both day and night (e.g., variable) and those that have matinal and crepuscular foraging were 146 classified as variable. We tested the association between color (pale and dark) and activity time 147 (diurnal, nocturnal, variable) using a Fisher's exact test [50]. 148 149 Body size and eye measurements 150 We measured body size and eye characteristics for workers from all 23 species listed 151 above. Body size was measured as mesosoma length, which is a standard measure for body size 152 in ants. Mesosoma length was measured as the diagonal length of the mesosoma in profile from 153 the point at which the pronotum meets the cervical shield to the posterior base of the metapleural 154 lobe. Mesosoma length was measured from photographs taken using a Spot Insight QE camera 155 attached to a Leica MZ 125 microscope. Images were then displayed on a video monitor, and 156 mesosoma length was measured using ImageJ (available at http://rsb.info.nih.gov/nih-image/). 157 Measurements were calibrated using photographs of an ocular micrometer scaled in 0.01 mm 158 increments.

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Eye measurements were made from high-resolution photographs of the left eye taken in 160 profile focused on the center of the eye at an angle that allowed viewing all facets. Photographs 161 were taken using a Leica M205C microscope at 100× that was linked to the stacking software 162 program Helicon Focus (www.heliconsoft.com/heliconsoft-products/helicon-focus). This 163 software combines photographs taken in different focus planes into one photograph where the 164 entire eye surface is in focus. Facet lenses were counted, and eye area and facet diameter (D) 165 were measured using Digimizer (https://www.digimizer.com/). The area tool was used to 166 calculate area. This tool also calculated the centroid of the eye, and D was the average of four 167 adjoining facets at the centroid. We also measured the diameter of the anterior ocellus for Briefly, a point was identified between two facets at the edge of the eye at its apex. We then 189 drew a line to a point on the eye surface two facet rows away in the anterior direction. This was 190 taken as the tangent to the eye at that point and the perpendicular bisector of these lines between 191 these points was drawn. This was also done for a line extending from the original point to a 192 point two facet rows in the posterior direction. The Δϕ was the angle between these two 193 perpendicular bisectors divided by two. We measured Δϕ three times in the same area for each 194 worker and used the average in our analysis. We calculated  for each worker as the product of 195 Δϕ in radians and D in m. 196 The same images that were used to measure Δϕ also were used to measure the anterior-197 posterior visual field span. Lines perpendicular to the tangent of the eye surface were drawn at 198 the anterior and posterior edge of each eye. The angular span of the visual field along the 199 anterior-posterior axis was characterized by the angle between these lines. This measurement 200 was repeated three times for each specimen and the mean value was used in our analyses. 201 202 Regional variation in D 203 Regional variation in D was measured for Myrmecocystus and Veromessor. The small 204 eyes of Aphaenogaster and Temnothorax contained too few facets to warrant examination of 205 11 regional variation. We quantified regional variation in D using the photographs taken for eye 206 size measurements (see above) for one pale and one dark species of Myrmecocystus (M. Diameter of the anterior ocellus (dependent variable) was compared similarly across species 222 (independent variable) of Myrmecocystus using analysis-of-covariance (ANCOVA). 223 Myrmecocystus navajo was omitted from this analysis because only one worker had a visible 224 anterior ocellus (see below). 225 Regional variation in D was analyzed using a one-way repeated measures ANOVA 226 followed by a LSD post-hoc test for each species [50]. The dependent variables -Δϕ, p, and 227 visual field span -were compared in separate ANCOVA's using genus (4 levels) and activity 12 period (diurnal versus nocturnal) as independent variables, with mesosoma length as a covariate 229 [50]. A Tukey's HSD post-hoc test compared differences across genera and species for each 230 variable. For all tests, data were transformed, as necessary, to meet the assumptions for 231 homogeneity of variance (Box's M test and Levene's test) and homogeneity of regression slopes. 232 233 Survey for additional pale ant species 234 We used Antweb (www.antweb.org) to scan photographs for pale ant species in the 235 genera Aphaenogaster, Crematogaster, Messor, and Temnothorax (subfamily Myrmicinae), and 236 Dorymyrmex and Iridomyrmex (subfamily Dolichoderinae). We scrolled through frontal 237 photographs of the head for all species in each of these genera looking for species that appeared 238 pale and that also appeared to have eyes that were larger than those of nearby dark species (e.g., 239 https://www.antweb.org/images.do?subfamily=myrmicinae&genus=temnothorax&rank=genus& 240 project=allantwebants). We verified our visual assessment of color for these taxa by measuring 241 their brightness value (B) using Adobe Photoshop, as detailed above. 242 243

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Pigmentation and daily activity pattern 245 We first quantitatively confirmed our visual impressions of variation in body color. As 246 predicted, our brightness values (B) measured from Antweb photographs were consistently and 247 significantly higher for species that we visually classified as pale compared to dark across all 248 four genera (t-test, t=-9.824 df, P < 0.0001). Mean B values were 76.3 (n = 10) for pale species 249 and 42.1 (n = 16) for dark species (Table 1, Fig 5). Values did not overlap for any pale and dark 250 species as all dark species had a mean B below 60, whereas all pale species had a mean B above 251 13 65. However, note that the two pale species of Veromessor displayed mean B values that ranged 252 from 65-70, which was intermediate to pale and dark species in the other three genera (Fig 5). 253 There also was a significant effect of color (pale, dark) on activity period (diurnal, nocturnal, 254 variable) (P < 0.0001, two-sided Fisher's exact test) with a preponderance of pale species that 255 are nocturnal and dark species that have diurnal/variable activity periods (Table 2) Temnothorax, but the Levene's test was significant for Veromessor (Table 3). We adjusted for 287 this effect by using a P value of 0.01 in our pairwise comparisons for species of Veromessor. 288 289    P < 0.001). All three eye features increased with body size within all six species (Fig 6). 328 The ANCOVA for diameter of the anterior ocellus was significant (Fig 7; tests  significantly lower than all other congeners (Fig 7). Note that M. navajo was not included in our 335 statistical analysis, but it was placed lowest in this group post-hoc because the anterior ocellus 336 was lacking in 11 of 12 workers. 337 20 Diameter of the anterior ocellus also increased with body size within all species except 338 M. navajo. Presence of the anterior ocellus also was associated with body size in M. mexicanus-339 01 and M. mexicanus-02, as workers with a mesosoma length < ≈ 2.2 mm lacked an anterior 340 ocellus while those with a mesosoma length > ≈ 2.2 mm had this ocellus, with ocellus diameter 341 increasing with body size in these latter workers (Fig 7). Both posterior ocelli usually were 342 present, but tiny, in workers that lacked an anterior ocellus.  (Table 4). 355 Mesosoma length was a significant covariate in the model (Wilks' λ = 0.502, F3,31 = 10.2, 356 P < 0.001), and tests of between-subjects effects were significant for eye area (F1,33 = 28.1, P < 357 0.001) and facet number (F1,33 = 17.1, P < 0.001), but not for mean D (F1,33 = 2.7, P = 0.11). 358 These patterns were evidenced in that eye area and facet number increased with body size within 359 all three species (Fig 8; A. boulderensis excluded because of small sample size), while mean D 360 21 increased with body size for A. megommata and A. patruelis, but it decreased with body size for 361 A. occidentalis (Fig 8).  (Table 4). 375 Mesosoma length was a significant covariate in the model (Wilks' λ = 0.448, F3,23 = 9.45, 376 P < 0.001), and tests of between-subjects effects were significant for eye area (F1,25 = 29.9, P < 377 0.001) and facet number (F1,25 = 20.7, P < 0.001), but not for mean D (F1,25 = 3.9, P = 0.058). 378 Eye area and facet number increased with body size within all three species, while mean D 379 increased with body size for T. sp. BCA-5 and T. tricarinatus, but it decreased with body size 380 for T. neomexicanus (Fig 9).  Fig 10). Based on the estimated marginal means, pairwise comparisons across 389 all species pairs using a LSD test showed that eye area was significantly larger for the pale V. 390 RAJ-pseu than for the dark V. smithi, and eyes for both species were significantly larger than the 391 pale V. lariversi. Eye area was significantly lower for all other dark congeners (Fig 10). Facet 392 number was significantly higher for V. RAJ-pseu than for V. smithi and V. lariversi, and these 393 three species were all significantly higher than all other dark congeners. Mean D (using 394 estimated marginal means) was highest for V. smithi and V. RAJ-pseu, followed by V. lariversi 395 and V. julianus, with the two latter species overlapping with V. RAJ-pseu but not V. smithi. 396 Mean D was significantly lower for all other dark congeners (P < 0.05, Fig 10; Table 4). Veromessor, and mean D increased for all species except V. smithi and V. chamberlini (Fig 10).

Visual field span 438
The ANCOVA for visual field span was significant for genus (F3,32 = 53.6, P < 0.001), 439 activity period (F1,32 = 151.7, P < 0.001), and the interaction of genus × activity period (F3,32 = 440 14.8, P < 0.001). Visual field span was greater for pale (mean = 98.8 o ) than for dark species 441 (mean = 73.0 o ), and the significant genus × activity period interaction indicated that differences 442 between the visual field of pale and dark species were larger in some genera, e.g., 443 Aphaenogaster, than others (Fig 13). Though not always significantly different, pale species had 444 a larger mean visual field in all four genera (Myrmecocystus t-test t8 df = -2.90, P = 0.10; 445 Aphaenogaster: t8 = 12.7, P < 0.001; Temnothorax: t-test t8 = -4.6, P = 0.002; Veromessor: t8 = -446 6.9, P < 0.001). The pale species of Myrmecocystus was also significantly different when 447 comparing the means when including mesosoma as a covariate (F1,7 = 87.0, P < 0.001). Across 448 genera the visual field was greatest for Myrmecocystus, intermediate for Aphaenogaster, and 449 smallest in Temnothorax and Veromessor (Tukey's HSD test, P < 0.05; Fig 13). 450 We also ran the above model with mesosoma length as a covariate, but it was not 451 significant (F1,31 = 2.6, P = 0.11), in part, because of the differing patterns exhibited across 452 25 species. For example, visual field span was positively correlated with mesosoma length in A. 453 megommata, A. occidentalis, T. BCA-5, T. neomexicanus, and V. lariversi, but these two 454 variables were negatively correlated in M. kennedyi, M. navajo, and V. chicoensis (Fig 13).  on available workers of these species, as detailed above, finding that numerous species displayed 484 a B value greater than 70, which we used as our lower threshold for pale species (Table 6). We 485 also visually judged that eyes for all of these species were larger than that of their dark 486 congeners. The combination of pale color and enlarged eyes occurred in numerous additional 487 species of Temnothorax from both the Old and New World, as well as in four additional genera -488 Crematogaster and Messor (subfamily Myrmicinae), and Dorymyrmex and Iridomyrmex 489 (subfamily Dolichoderinae) (Table 6); the latter two genera comprise a third subfamily 490 containing pale species. Moreover, this combination of traits occurred in both the Old and New 491 World, and they were especially common in Temnothorax (Table 6), where these traits evolved 492 independently in multiple species groups (in at least two species groups in the United States and 493 Mexico (T. silvestrii and T. tricarinatus) and in at least one species group in northern Africa (T. 494 laciniatus) (Table 6)  Foraging and cuticular color 512 Worker color was correlated with foraging time across all four genera of ants, suggesting 513 that pale coloration is linked to nocturnal foraging in these and other ants. Alternatively, dark 514 species usually forage diurnally, but some species also forage nocturnally during warm seasons, 515 and several species are largely matinal-crepuscular-nocturnal foragers. Moreover, pale color 516 involves repeated evolution of similar color phenotypes in response to living in dim light to 517 lightless environments both in these ants and in numerous other organisms [13, 14, 24], but it is 518 not a necessary phenotype given the numerous taxa living in similarly dim conditions that have 519 retained their pigmentation. 520 A species-level phylogeny is available for all four genera such that we can infer the 521 direction of trait evolution. These phylogenies infer that pale color is a derived trait in 522  Table 4), about 1.3-1.5-fold higher for A. megommata compared 547 to its dark congeners, and about 1.5-1.7-fold higher for T. sp. BCA-5 compared to its dark 548 congeners. Alternatively, for Veromessor, facet area was highest in the dark V. smithi. The 549 mean difference was about 1.05-1.10× higher for V. smithi than for V. lariversi and V. RAJ-550 pseu; all other dark congeners had a smaller D. This study examined only D, suggesting that 551 these sensitivity values are minimum differences between pale and dark species. Pale species of 552 Myrmecocytus also differed in that their eyes were more protruding and dome-shaped compared 553 to the more flattened eyes of their dark congeners (Fig 1). These more bulging, dome-shaped 554 eyes result in a greater radius of curvature and possibly a greater visual span field, as well as 555 space for more facets within a given eye area. 556 Interestingly, the two pale sister species of Veromessor, V. RAJ-pseu and V. lariversi, 557 displayed different patterns of eye structure, with V. RAJ-pseu having larger eyes and more 558 facets than V. lariversi (Fig 9). Additionally, eyes of the dark species V. smithi were smaller 559 with fewer facets than V. RAJ-pseu, but they were larger with larger D's compared to V. 560 lariversi. This may result from the fact that V. smithi is the most nocturnally-active of all dark 561 species in the genus. One difference between V. lariversi and V. RAJ-pseu and other pale 562 species examined herein is their more yellowish-amber to yellowish-orange color and lower B 563 value, indicating that they are less pale than pale species in the other three genera (see Table 1; 564 Figs 1-4). Differences in eye structure across genera along with variation across species of 565 Veromessor are similar to the wide variation in degree of pigment loss and eye degeneration 566 found among cave-dwelling species that is caused by differences in divergence time and 567 intensity of selection [16]. Similar variation occurs for nocturnal foraging bees in which many 568 species have a relatively pale body color, and many but not all species have enlarged compound 569 eyes and ocelli [18]. 570 All of our species had relatively large Δϕ's that ranged from 3.5-7 o . Pale and dark 571 species varied in their patterns of Δϕ which were significantly larger in the dark Temnothorax 572 and Aphaenogaster, which had small eyes with fewer facets, but was larger for the pale 573 Myrmecocystus which had numerous eye facets. Moreover, Δϕ did not decrease for pale species 574 indicating that daily activity patterns have had little effect on the evolution of resolving power. 575 The eye parameter () measures the tradeoff between sensitivity and resolution, with eyes 576 that require higher sensitivity having larger  values. Consequently, insects active during high 577 light conditions usually have low  values that enhance resolution, whereas species active in low 578 light have higher  values that often exceed 2 um rad [34]. Across our four genera,  was 579 32 significantly higher only for the pale M. navajo compared to the dark M. kennedyi, with  for the 580 former species approaching 2 (Fig 11). The higher  value for M. navajo resulted from the 581 combination of significantly larger facets and a significantly larger Δϕ (Figs 5 & 10). In 582 contrast, M. kennedyi was the only strictly diurnal forager among all dark species (Table 1), and 583 correspondingly it had the lowest mean  value (0.91) among all species (Fig 11). The  value 584 was similar for the other three pairs of congeners, with the dark A. occidentalis having the 585 highest  value (2.12) of all species (Fig 10). The lack of significant and consistent patterns 586 across the latter three genera likely reflect the wide range of light conditions under which dark 587 species forage including nocturnal foraging in some seasons (Table 1). 588 The visual field was larger, usually significantly so, for pale species in all four genera 589 (Fig 13). Moreover, there was no indication that these species had binocular vision in the 590 anterior-posterior direction, that is, they cannot use their eyes for binocular depth perception. 591 This infers that these ants do not use vision to find or capture food items, which aligns with diets 592 that include stationary objects such as seeds, dead insects, and extrafloral nectaries. Instead, it 593 seems likely their eyes are used for detection and orientation relative to land-based and celestial 594 cues used in navigation (see below). In addition, our finding that visual field usually correlated 595 with body size (positively or negatively, depending on the species), contrasted with the pattern 596 for Cataglyphis bicolor, in which visual field was independent of body size [66]. 597 Regional variation in D is common in insects [67], with these size differences probably 598 related to the different selection pressures on eye structure in each region. Larger facets imply 599 that insects have better vision from regions containing larger facets. In this study, ventral facets 600 were significantly larger in three of the four examined species (along with anterior facets in two 601 species), and they also were largest in the fourth species (M. kennedyi) but the difference was 602      (see text and Table 1, Figs 1-4).   occidentalis, and A. patruelis are dark (filled symbols and bold font) (see text). For each species, number of workers examined and number of colonies they were derived from is given in parentheses. Significant differences (P < 0.05) among species are denoted after each species name by the letters a-c: a > b > c; the three sets of letters for each species correspond to panels A, B, and C, respectively. Groupings are based on univariate F tests within MANCOVA using the estimated marginal means followed by pairwise comparisons using a least significant differences test (see text). Foraging time for each species is given in Table 1. triacarinatus are dark (filled symbols and bold font) (see text). For each species, number of workers examined and number of colonies they were derived from is given in parentheses.
Significant differences (P < 0.05) among species are denoted after each species name by the letters a-b: a > b; the three sets of letters for each species correspond to panels A, B, and C, respectively. Groupings are based on univariate F tests within MANCOVA using the estimated marginal means followed by pairwise comparisons using a least significant differences test (see text). Foraging time for each species is given in Table 1. b > c > d > e > f > g; the three sets of letters for each species correspond to panels A, B, and C, respectively. Groupings are based on univariate F tests within MANCOVA using the estimated marginal means followed by pairwise comparisons using a least significant differences test (see text). Foraging time for each species is given in Table 1. the same x-axis and y-axis scaling in order to visualize differences between light and dark species across genera. Mean Δϕ (in degrees) is given after each species name with an asterisk denoting the species with a significant larger Δϕ based on a t-test (P < 0.05). The significant interaction of genus × activity period is shown by larger Δϕ's for pale species of Myrmecocystus and Veromessor, whereas Δϕ was larger for dark species of Aphaenogaster and Temnothorax.
Sample size is n = 5 for each species. the same x-axis and y-axis scaling in order to visualize differences between light and dark species across genera. Mean p is given after each species name with an asterisk denoting the species with a significant larger p based on a t-test (P < 0.05). The significant interaction of genus × activity period is shown by larger differences between light-colored and dark-colored species of Aphaenogaster compared to those in the other three genera. Sample size is n = 5 for each species. the same x-axis and y-axis scaling in order to visualize differences between pale and dark species across genera. Mean visual field span (in degrees) is given after each species name with an asterisk denoting the species with a significant larger visual field based on a t-test (P < 0.05); the double asterisk denotes that the t-test was not significant, but that the visual field was significantly larger when including mesosoma length as a covariate. The significant interaction of genus × activity period is shown by larger differences between pale and dark species of Aphaenogaster compared to those in the other three genera. Sample size is n = 5 for each species. for each species are based on a repeated-measures ANOVA followed by a least significant